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Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

Diaz-Sylvester PL, Porta M, Copello JA - PLoS ONE (2011)

Bottom Line: To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers.However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)).In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Southern Illinois School of Medicine, Springfield, Illinois, United States of America.

ABSTRACT
Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

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Effects of M2+ added to the luminal surface of RyR2.(A) Luminal M2+ alters open probability of Ca2+-activated RyR2. Representative recordings of RyR exposed to increasing concentration of cytosolic Ca2+, in the presence of luminal Ba2+ (left panel) or Mg2+ (middle panel). Mean Po as a function of cytosolic Ca2+ observed in RyR2 bathed with different luminal cations (right panel). Maximal open probability values of channels exposed to 50 mM luminal M2+ were higher than those observed with 100 mM luminal Cs+ (p<0.05). The EC50 for cytosolic Ca2+ activation in the presence of luminal Mg2+ was 10.4±0.6 µM (n = 4). This is substantially higher than the EC50 with luminal Cs+, Ca2+ or Sr2+ (3.2±0.3 µM, n = 5; 2.9±0.3, n = 7; and 3.4±0.4 µM, n = 5 respectively). EC50 with Ba2+ was intermediate (6.7±0.3 µM, n = 7). (B) Luminal M2+ effect is more evident in the presence of caffeine. Representative recordings of RyR exposed to increasing concentration of cytosolic caffeine, in the presence of luminal Ca2+ (left panel) or Ba2+ (middle panel). The left panel shows mean Po as a function of cytosolic caffeine observed in RyR2 bathed with different luminal M2+ (n = 5 in each condition). Recordings were made at 100 nM cytosolic free Ca2+. In the presence of luminal Ca2+ or Sr2+, caffeine activated RyR2 to high Po (EC50's were 5.8±0.5 and 6.4±0.4 mM respectively). With luminal Ba2+ or Mg2+, RyR2 poorly activated or remained closed. (C) Luminal M2+ affects the gating of caffeine-activated RyR2. Left panels: representative single channel recordings. Right panels: Open and closed dwell-time distribution histograms of caffeine-activated RyR2 bathed with different luminal M2+. All recordings were made at a holding potential of 0 mV. The cytosolic solutions contained 100 nM cytosolic Ca2+ and 20 mM caffeine. Dwell open (τo) and closed (τc) times with different luminal M2+ were obtained by fitting the logarithmic dwell time distributions (open or close events distributions) with two components. Openings with luminal Ca2+ distributed with τo = 154 ± 1 ms (100% events) while closures distributed with τc1 = 1.00 ± 0.21 ms (17% of the events) and τc2 = 143 ± 9 ms (83%). Values with Sr2+ were τo1 = 1.32 ± 0.14 ms (18%), τo2 = 114 ± 5 ms (82%), τc1 = 2.2 ± 0.3 ms (28%) and τc2 = 23.0 ± 2.5 ms (72%). Values with Ba2+ were τo = 0.6 ± 0.1 ms (100%), τc1 = 1.3 ± 0.1 ms (37%) and τc2 = 23.0 ± 1.1 (63%). Values with Mg2+ were τo = 0.8 ± 0.1 ms (100%), τc1 = 0.5 ± 0.1 ms (43%) and τc2 = 11.2 ± 1.2 (57%).
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pone-0026693-g002: Effects of M2+ added to the luminal surface of RyR2.(A) Luminal M2+ alters open probability of Ca2+-activated RyR2. Representative recordings of RyR exposed to increasing concentration of cytosolic Ca2+, in the presence of luminal Ba2+ (left panel) or Mg2+ (middle panel). Mean Po as a function of cytosolic Ca2+ observed in RyR2 bathed with different luminal cations (right panel). Maximal open probability values of channels exposed to 50 mM luminal M2+ were higher than those observed with 100 mM luminal Cs+ (p<0.05). The EC50 for cytosolic Ca2+ activation in the presence of luminal Mg2+ was 10.4±0.6 µM (n = 4). This is substantially higher than the EC50 with luminal Cs+, Ca2+ or Sr2+ (3.2±0.3 µM, n = 5; 2.9±0.3, n = 7; and 3.4±0.4 µM, n = 5 respectively). EC50 with Ba2+ was intermediate (6.7±0.3 µM, n = 7). (B) Luminal M2+ effect is more evident in the presence of caffeine. Representative recordings of RyR exposed to increasing concentration of cytosolic caffeine, in the presence of luminal Ca2+ (left panel) or Ba2+ (middle panel). The left panel shows mean Po as a function of cytosolic caffeine observed in RyR2 bathed with different luminal M2+ (n = 5 in each condition). Recordings were made at 100 nM cytosolic free Ca2+. In the presence of luminal Ca2+ or Sr2+, caffeine activated RyR2 to high Po (EC50's were 5.8±0.5 and 6.4±0.4 mM respectively). With luminal Ba2+ or Mg2+, RyR2 poorly activated or remained closed. (C) Luminal M2+ affects the gating of caffeine-activated RyR2. Left panels: representative single channel recordings. Right panels: Open and closed dwell-time distribution histograms of caffeine-activated RyR2 bathed with different luminal M2+. All recordings were made at a holding potential of 0 mV. The cytosolic solutions contained 100 nM cytosolic Ca2+ and 20 mM caffeine. Dwell open (τo) and closed (τc) times with different luminal M2+ were obtained by fitting the logarithmic dwell time distributions (open or close events distributions) with two components. Openings with luminal Ca2+ distributed with τo = 154 ± 1 ms (100% events) while closures distributed with τc1 = 1.00 ± 0.21 ms (17% of the events) and τc2 = 143 ± 9 ms (83%). Values with Sr2+ were τo1 = 1.32 ± 0.14 ms (18%), τo2 = 114 ± 5 ms (82%), τc1 = 2.2 ± 0.3 ms (28%) and τc2 = 23.0 ± 2.5 ms (72%). Values with Ba2+ were τo = 0.6 ± 0.1 ms (100%), τc1 = 1.3 ± 0.1 ms (37%) and τc2 = 23.0 ± 1.1 (63%). Values with Mg2+ were τo = 0.8 ± 0.1 ms (100%), τc1 = 0.5 ± 0.1 ms (43%) and τc2 = 11.2 ± 1.2 (57%).

Mentions: It has been reported that RyR2 channel function can also be regulated by luminal Ca2+ [13], [14], [20], [25], [26]. Fewer studies have explored the sensitivity of these luminal sites to different divalent cations [13], [15]. Figure 2A shows RyR2 sensitivity to cytosolic Ca2+ with different luminal cations (Ca2+, Sr2+, Mg2+, Ba2+ and Cs+). In these experiments, the luminal cations were also the charge carriers (the current flows in lumen-to-cytosol direction). With luminal Cs+, Ca2+ or Sr2+, the channels were activated by cytosolic Ca2+ with an EC50 ∼ 3 µM (Fig. 2A). In contrast, significantly higher cytosolic Ca2+ levels were required to activate RyR2 in the presence of luminal Ba2+ or Mg2+ (EC50 ∼ 6.7 µM and 10.4 µM, respectively). At high cytosolic Ca2+ levels (>100 µM), the maximal Po (plateau) value was similar in the presence of luminal Ca2+, Sr2+, Mg2+ or Ba2+. However, this value was significantly reduced (from Po ∼0.8 to Po ∼0.4) when we used Cs+ as charge carrier (luminal solution contained 100 mM Cs+ plus ∼500 µM luminal Ca2+). Open probabilities were much lower with luminal Cs+ solutions (where only micromolar contaminant Ca2+ was present) , but Po at 0 mV cannot be accurately determined using this approach because the channels are prone to inactivation (results not shown). In Supporting Information (Fig. S1) we show that cytosolic Ca2+ increases Po of RyR2 bathed with luminal Ca2+ by increasing the number of openings and by increasing event duration. A similar pattern (albeit with overal briefer events) is observed when RyR2 are bathed with luminal Sr2+, Mg2+ and Ba2+.


Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

Diaz-Sylvester PL, Porta M, Copello JA - PLoS ONE (2011)

Effects of M2+ added to the luminal surface of RyR2.(A) Luminal M2+ alters open probability of Ca2+-activated RyR2. Representative recordings of RyR exposed to increasing concentration of cytosolic Ca2+, in the presence of luminal Ba2+ (left panel) or Mg2+ (middle panel). Mean Po as a function of cytosolic Ca2+ observed in RyR2 bathed with different luminal cations (right panel). Maximal open probability values of channels exposed to 50 mM luminal M2+ were higher than those observed with 100 mM luminal Cs+ (p<0.05). The EC50 for cytosolic Ca2+ activation in the presence of luminal Mg2+ was 10.4±0.6 µM (n = 4). This is substantially higher than the EC50 with luminal Cs+, Ca2+ or Sr2+ (3.2±0.3 µM, n = 5; 2.9±0.3, n = 7; and 3.4±0.4 µM, n = 5 respectively). EC50 with Ba2+ was intermediate (6.7±0.3 µM, n = 7). (B) Luminal M2+ effect is more evident in the presence of caffeine. Representative recordings of RyR exposed to increasing concentration of cytosolic caffeine, in the presence of luminal Ca2+ (left panel) or Ba2+ (middle panel). The left panel shows mean Po as a function of cytosolic caffeine observed in RyR2 bathed with different luminal M2+ (n = 5 in each condition). Recordings were made at 100 nM cytosolic free Ca2+. In the presence of luminal Ca2+ or Sr2+, caffeine activated RyR2 to high Po (EC50's were 5.8±0.5 and 6.4±0.4 mM respectively). With luminal Ba2+ or Mg2+, RyR2 poorly activated or remained closed. (C) Luminal M2+ affects the gating of caffeine-activated RyR2. Left panels: representative single channel recordings. Right panels: Open and closed dwell-time distribution histograms of caffeine-activated RyR2 bathed with different luminal M2+. All recordings were made at a holding potential of 0 mV. The cytosolic solutions contained 100 nM cytosolic Ca2+ and 20 mM caffeine. Dwell open (τo) and closed (τc) times with different luminal M2+ were obtained by fitting the logarithmic dwell time distributions (open or close events distributions) with two components. Openings with luminal Ca2+ distributed with τo = 154 ± 1 ms (100% events) while closures distributed with τc1 = 1.00 ± 0.21 ms (17% of the events) and τc2 = 143 ± 9 ms (83%). Values with Sr2+ were τo1 = 1.32 ± 0.14 ms (18%), τo2 = 114 ± 5 ms (82%), τc1 = 2.2 ± 0.3 ms (28%) and τc2 = 23.0 ± 2.5 ms (72%). Values with Ba2+ were τo = 0.6 ± 0.1 ms (100%), τc1 = 1.3 ± 0.1 ms (37%) and τc2 = 23.0 ± 1.1 (63%). Values with Mg2+ were τo = 0.8 ± 0.1 ms (100%), τc1 = 0.5 ± 0.1 ms (43%) and τc2 = 11.2 ± 1.2 (57%).
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pone-0026693-g002: Effects of M2+ added to the luminal surface of RyR2.(A) Luminal M2+ alters open probability of Ca2+-activated RyR2. Representative recordings of RyR exposed to increasing concentration of cytosolic Ca2+, in the presence of luminal Ba2+ (left panel) or Mg2+ (middle panel). Mean Po as a function of cytosolic Ca2+ observed in RyR2 bathed with different luminal cations (right panel). Maximal open probability values of channels exposed to 50 mM luminal M2+ were higher than those observed with 100 mM luminal Cs+ (p<0.05). The EC50 for cytosolic Ca2+ activation in the presence of luminal Mg2+ was 10.4±0.6 µM (n = 4). This is substantially higher than the EC50 with luminal Cs+, Ca2+ or Sr2+ (3.2±0.3 µM, n = 5; 2.9±0.3, n = 7; and 3.4±0.4 µM, n = 5 respectively). EC50 with Ba2+ was intermediate (6.7±0.3 µM, n = 7). (B) Luminal M2+ effect is more evident in the presence of caffeine. Representative recordings of RyR exposed to increasing concentration of cytosolic caffeine, in the presence of luminal Ca2+ (left panel) or Ba2+ (middle panel). The left panel shows mean Po as a function of cytosolic caffeine observed in RyR2 bathed with different luminal M2+ (n = 5 in each condition). Recordings were made at 100 nM cytosolic free Ca2+. In the presence of luminal Ca2+ or Sr2+, caffeine activated RyR2 to high Po (EC50's were 5.8±0.5 and 6.4±0.4 mM respectively). With luminal Ba2+ or Mg2+, RyR2 poorly activated or remained closed. (C) Luminal M2+ affects the gating of caffeine-activated RyR2. Left panels: representative single channel recordings. Right panels: Open and closed dwell-time distribution histograms of caffeine-activated RyR2 bathed with different luminal M2+. All recordings were made at a holding potential of 0 mV. The cytosolic solutions contained 100 nM cytosolic Ca2+ and 20 mM caffeine. Dwell open (τo) and closed (τc) times with different luminal M2+ were obtained by fitting the logarithmic dwell time distributions (open or close events distributions) with two components. Openings with luminal Ca2+ distributed with τo = 154 ± 1 ms (100% events) while closures distributed with τc1 = 1.00 ± 0.21 ms (17% of the events) and τc2 = 143 ± 9 ms (83%). Values with Sr2+ were τo1 = 1.32 ± 0.14 ms (18%), τo2 = 114 ± 5 ms (82%), τc1 = 2.2 ± 0.3 ms (28%) and τc2 = 23.0 ± 2.5 ms (72%). Values with Ba2+ were τo = 0.6 ± 0.1 ms (100%), τc1 = 1.3 ± 0.1 ms (37%) and τc2 = 23.0 ± 1.1 (63%). Values with Mg2+ were τo = 0.8 ± 0.1 ms (100%), τc1 = 0.5 ± 0.1 ms (43%) and τc2 = 11.2 ± 1.2 (57%).
Mentions: It has been reported that RyR2 channel function can also be regulated by luminal Ca2+ [13], [14], [20], [25], [26]. Fewer studies have explored the sensitivity of these luminal sites to different divalent cations [13], [15]. Figure 2A shows RyR2 sensitivity to cytosolic Ca2+ with different luminal cations (Ca2+, Sr2+, Mg2+, Ba2+ and Cs+). In these experiments, the luminal cations were also the charge carriers (the current flows in lumen-to-cytosol direction). With luminal Cs+, Ca2+ or Sr2+, the channels were activated by cytosolic Ca2+ with an EC50 ∼ 3 µM (Fig. 2A). In contrast, significantly higher cytosolic Ca2+ levels were required to activate RyR2 in the presence of luminal Ba2+ or Mg2+ (EC50 ∼ 6.7 µM and 10.4 µM, respectively). At high cytosolic Ca2+ levels (>100 µM), the maximal Po (plateau) value was similar in the presence of luminal Ca2+, Sr2+, Mg2+ or Ba2+. However, this value was significantly reduced (from Po ∼0.8 to Po ∼0.4) when we used Cs+ as charge carrier (luminal solution contained 100 mM Cs+ plus ∼500 µM luminal Ca2+). Open probabilities were much lower with luminal Cs+ solutions (where only micromolar contaminant Ca2+ was present) , but Po at 0 mV cannot be accurately determined using this approach because the channels are prone to inactivation (results not shown). In Supporting Information (Fig. S1) we show that cytosolic Ca2+ increases Po of RyR2 bathed with luminal Ca2+ by increasing the number of openings and by increasing event duration. A similar pattern (albeit with overal briefer events) is observed when RyR2 are bathed with luminal Sr2+, Mg2+ and Ba2+.

Bottom Line: To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers.However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)).In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Southern Illinois School of Medicine, Springfield, Illinois, United States of America.

ABSTRACT
Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

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Related in: MedlinePlus